PROJECT SUMMARY Efforts to develop an effective vaccine against HIV-1/AIDS continue to be hampered by an incomplete understanding of the immune responses needed for protection. Non-neutralizing antibody functions, such as the elimination of virus-infected cells by antibody-dependent cellular cytotoxicity (ADCC), are widely believed to have contributed to the reduced risk of HIV-1 infection among vaccine recipients in the RV144 trial; however, the antibody specificities and effector functions underlying this protection have not been clearly defined. This uncertainty can be attributed in part to differences in methods for measuring ADCC, a limited understanding of the factors influencing the sensitivity of HIV-infected cells to antibodies, and a lack of definitive evidence for protection by non-neutralizing antibodies in animal models. Although correlative evidence suggests that antibodies to variable 1 and 2 (V1V2) region and CD4-inducible (CD4i) epitopes of HIV-1 gp120 may have contributed to protection, this has been difficult to confirm experimentally. The proposed studies will therefore take advantage of the combined expertise of our research team to improve methods for measuring ADCC and to test the antibody specificities and effector functions implicated in the outcome of the RV144 trial in a nonhuman primate model. In Aim 1, we will determine the impact of viral and cellular factors that modulate the sensitivity of HIV-infected cells to antibodies on methods for measuring ADCC. In Aim 2, we will develop and implement a high-throughput assay platform to enable a comprehensive assessment of ADCC directed against distinct conformations of the HIV-1 envelope glycoprotein. In Aim 3, we will test the hypothesis that V1V2- and/or CD4i-specific antibodies can afford partial protection in a low-dose mucosal SHIV challenge model designed to simulate conditions of HIV-1 CRF01_AE transmission in Thailand, where the RV144 trial took place. In Aim 4, we will test Fc domain variants of V1V2- and CD4i-specific antibodies for the ability to protect macaques against low-dose mucosal SHIV challenge to determine the extent to which Fc-mediated effector functions may contribute to protection. These unprecedented studies are expected to reveal fundamental molecular mechanisms that influence the sensitivity of HIV-infected cells to antibodies and their implications for measuring ADCC, provide a standardizable, high-throughput assay for quantifying ADCC as a correlate of protection, and yield greater insight into the types of antibody responses that may ultimately be needed for protection against HIV-1.